Robert Wattiaux

Endosomes, lysosomes: their implication in gene transfer

Plasmid DNA, naked or bound to a non-viral vector, is taken up by endocytosis. As a result, it has to travel through the intracellular endocytic pathway involving endosomes and lysosomes. However, some DNA molecules must escape these organelles to reach the nucleus where transcription takes place. In this paper, we consider different factors that could affect the trafficking of plasmid DNA and influence transfection efficiency.

Expression of Lamp‐1 and Lamp‐2 and their interactions with galectin‐3 in human tumor cells

Lysosomal‐membrane‐associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell‐surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E‐selectin and between Lamps and galectins (endogenous‐galactoside‐binding lectins). The present study examined the expression of Lamp‐1 and Lamp‐2 and their interactions with galectin‐3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell‐surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo‐2 (human colon‐adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin‐3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin‐3 binding to Lamps‐expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis. Int. J. Cancer 75:105–111, 1998.© 1998 Wiley‐Liss, Inc.

Cationic lipids destabilize lysosomal membrane in vitro

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N‐(1‐(2,3‐dioleoxy)propyl)‐N,N,N,‐trimethylammonium methyl‐sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of β‐galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection.

The mechanism of gene transfer into hepatocytes by the hydrodynamics‐based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection.

Deleterious effects of xanthine oxidase on rat liver endothelial cells after ischemia/reperfusion

Previous studies have demonstrated that reactive oxygen species are involved in ischemic injury. The present work was undertaken to determine in vivo the role of xanthine oxidase in the oxygen free radical production during rat liver ischemia and to examine the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) during the same period. Our results indicate a 4-fold increase in xanthine oxidase activity between 2 and 3 hours of normothermic ischemia, in parallel with a decrease in cell viability. Moderate hypothermia delays both events. Under the same conditions, the activity of oxygen radical scavenging enzymes remains unchanged. Moreover, we have compared in vitro the susceptibility of isolated liver cells to an oxidative stress induced by O2.-, H2O2 and .OH. Our results reveal that endothelial cells are much more susceptible to reactive oxygen species than hepatocytes, probably because they lack H2O2-detoxifying enzymes. These findings suggest that xanthine oxidase might play a major role in the ischemic injury mainly at the level of the sinusoidal space where most endothelial cells are located.

Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume.

An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (hydrodynamics‐based transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA.

Effect of various flavonoids on lysosomes subjected to an oxidative or an osmotic stress.

When a light mitochondrial fraction (L fraction) of rat liver is incubated in the presence of an oxygen free radical generating system (xanthine-xanthine oxidase), the free activity of N-acetylglucosaminidase (NAGase) increases as a result of the deterioration of the lysosomal membrane. Various flavonoids are able to prevent this phenomenon, others are ineffective. Comparative activity studies suggest the importance of the presence of two OH groups in orthosubstitution in the B ring and of an OH in the 3 position. Flavan-type flavonoids behave like their related flavonoids; d-catechin also opposes lysosome disruption. Kaempferol, quercetin, 7,8-dihydroxyflavone and d-catechin inhibit lipoperoxidation occurring in an L fraction incubated with the xanthine oxidase system as ascertained by malondialdehyde (MDA) production. For kaempferol and quercetin, such an inhibition parallels the prevention of NAGase release; this is not the case for the two other compounds where inhibition of NAGase release takes place at a flavonoid concentration lower than that required to oppose MDA production. Morphological observations performed on purified lysosomes confirm the biochemical results. Some flavonoids are also able to prevent release of NAGase caused by the incubation of an L fraction in isoosmotic glucose. Only flavone and hydroxyflavones are effective. It is proposed that the protective effect of flavonoids on lysosomes subjected to oxygen free radicals does not only originate from their scavenger and antilipoperoxidant properties; a more direct action on lysosomal membrane making it more resistant to oxidative aggression has to be considered. The prevention by some flavonoids of lysosome osmotic disruption in isoosmotic glucose could be the result of an inhibition of glucose translocation through the lysosomal membrane.

Uptake by rat liver and intracellular fate of plasmid DNA complexed with poly-l-lysine or poly-d-lysine

Efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. When a non‐viral vector is used, it is not known to what extent the plasmid DNA catabolism is subordinated to the catabolism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [35S]DNA complexed with a cationic peptide poly‐l‐lysine that can be hydrolyzed by cellular peptidases or with its stereoisomer, poly‐d‐lysine, that cannot be split by these enzymes. Complexes of DNA with poly‐l‐lysine and poly‐d‐lysine are taken up to the same extent by the liver, mainly by Kupffer cells, but the intracellular degradation of nucleic acid molecules is markedly quicker when poly‐l‐lysine is injected. The association of DNA with the polycations inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly‐l‐lysine and poly‐d‐lysine. The intracellular journey followed by [35S]DNA complexed with poly‐l‐ or poly‐d‐lysine was investigated using differential and isopycnic centrifugation. Results indicate that [35S]DNA is transferred more slowly to lysosomes, the main site of intracellular degradation of endocytosed macromolecules, when it is given as a complex with poly‐d‐lysine than with poly‐l‐lysine. They suggest that the digestion of the vector in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes. Such a phenomenon could explain why injected plasmid DNA is more stable in vivo when it is associated with poly‐d‐lysine.

Lateral phase separations and structural integrity of the inner membrane of rat-liver mitochondria: Effect of compression. Implications in the centrifugation of these organelles

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium. Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0 degrees C, when the pressure reaches 750 kg/cm2. Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal lead to gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes. These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.

Subcellular Localization of Mannose 6-Phosphate Glycoproteins in Rat Brain

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification ofN-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.